玉米MYB家族ZmMYB3R基因的克隆及抗逆功能研究

发布时间：2018-09-18 07:50

【摘要】：玉米作为重要的粮食作物和工业原料,在其生物进化过程中为应对各种逆境胁迫形成了复杂的调控网络。其中,通过转录因子与靶基因的结合来调控各种逆境相关基因的表达,可以提高植物对逆境的抗性。研究表明,MYB家族转录因子能够积极地响应植物逆境胁迫。其中R2R3亚家族是高度保守的、最为广泛的一类参与植物逆境胁迫的亚家族。而R1R2R3亚家族基因在植物逆境胁迫中的功能报道还较少,只有个别基因在拟南芥和水稻以及小麦中被发现与耐盐、耐旱等一些抗逆功能相关。本研究通过生物信息学分析并筛选了玉米中R1R2R3亚家族基因,结合PCR方法从玉米中克隆了Zm MYB3R基因。利用亚细胞定位、荧光定量PCR以及过表达转基因手段等一系列研究方法明确了该基因在玉米抗逆过程中的功能,并对其响应逆境胁迫的机制做了初步分析。具体结果如下:1.序列分析发现Zm MYB3R基因全长为1692bp,编码563个氨基酸,分子质量为61.66k D,等电点为8.33。在N端有三个MYB结构域,每个结构域中有三个色氨酸残基。同时Zm MYB3R基因与近缘物种逆境相关的3R基因的进化关系非常紧密。2.荧光定量PCR结果表明Zm MYB3R基因受外源ABA、干旱和高盐胁迫条件的诱导表达。同时组织表达模式分析显示Zm MYB3R在各个组织中均有不同程度的表达,其中在茎和叶片中表达量较高。3.通过亚细胞定位分析发现,Zm MYB3R转录因子是核定位蛋白。转录活性分析表明该基因具有转录活性,通过筛选得出活性区域为217-563bp。4.利用PCR方法构建过表达载体结合农杆菌侵染法获得转基因拟南芥植株。在MS培养基上添加Na Cl和Mannitol模拟高盐、干旱环境。除此之外在真实环境下高盐和干旱处理植株,并测定伤害度。胁迫处理结果显示:相较于野生型,转基因植株的耐盐和抗旱能力明显提高。5.ABA敏感性实验表明,转基因植株种子的萌发率明显高于野生型,叶片气孔孔径显著减小。用外源的ABA处理转基因型和野生型的拟南芥,发现转基因型的拟南芥的发芽率明显高于野生型。而转基因拟南芥的气孔在ABA处理后孔径相对变的更窄。6.荧光定量PCR分析显示转基因植株中的ABA合成及下游相关基因的表达较野生型明显上调,表明Zm MYB3R通过调控ABA信号途径增强植物的抗逆性。说明Zm MYB3R可以依赖于ABA诱导下游逆境相关基因表达,从而提高植物的非生物胁迫的耐受力。
[Abstract]:Maize, as an important food crop and industrial raw material, has formed a complex regulatory network in the course of its biological evolution to cope with various stresses. Among them, the expression of various stress-related genes can be regulated by the combination of transcription factors and target genes, which can improve the resistance of plants to stress. The results showed that MYB family transcription factors could respond positively to plant stress. Among them, R2R3 subfamily is highly conserved and most widely involved in plant stress. However, the function of R1R2R3 subfamily genes in plant stress was less reported. Only a few genes were found in Arabidopsis, rice and wheat, which were related to salt tolerance, drought tolerance and so on. In this study, R1R2R3 subfamily genes in maize were analyzed and screened by bioinformatics, and Zm MYB3R gene was cloned from maize by PCR method. A series of research methods, such as subcellular localization, fluorescence quantitative PCR and over-expression transgenic methods, were used to clarify the function of the gene in maize stress resistance, and the mechanism of its response to stress was preliminarily analyzed. The results are as follows: 1. Sequence analysis showed that the Zm MYB3R gene was 1692 BP, encoding 563 amino acids, with molecular weight of 61.66 KD and isoelectric point of 8.33. There are three MYB domains at the N terminal and three tryptophan residues in each domain. At the same time, the evolutionary relationship between Zm MYB3R gene and the 3R gene of relative species is very close. The results of fluorescence quantitative PCR showed that the expression of Zm MYB3R gene was induced by exogenous ABA, drought and high salt stress. At the same time, tissue expression pattern analysis showed that Zm MYB3R was expressed in different degree in all tissues, especially in stem and leaf. Subcellular localization analysis showed that Zm MYB3R transcription factor was a nuclear localization protein. The transcriptional activity analysis showed that the gene had transcriptional activity, and the active region was 217-563bp.4. Transgenic Arabidopsis thaliana plants were obtained by using PCR method to construct overexpression vector combined with Agrobacterium tumefaciens infection. Na Cl and Mannitol were added to MS medium to simulate high salt and dry environment. In addition, the plants were treated with high salt and drought in real environment, and the degree of injury was measured. The results of stress treatment showed that the sensitivity of transgenic plants to salt tolerance and drought resistance was significantly higher than that of wild type. The results of ABA sensitivity test showed that the germination rate of transgenic plants was significantly higher than that of wild type, and the stomatal aperture of leaves was significantly decreased. Transgenic type and wild type Arabidopsis thaliana were treated with exogenous ABA. It was found that the germination rate of transgenic type Arabidopsis thaliana was significantly higher than that of wild type. The pore diameter of transgenic Arabidopsis thaliana was narrower than that of ABA treatment. Fluorescence quantitative PCR analysis showed that ABA synthesis and downstream gene expression in transgenic plants were significantly up-regulated than those in wild type, indicating that Zm MYB3R enhanced plant stress resistance by regulating ABA signaling pathway. It was suggested that Zm MYB3R could induce downstream stress-related gene expression by ABA, thus enhancing plant abiotic stress tolerance.
【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2;S513

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